Not applicable.
The present invention pertains generally to method and apparatus for separating and concentrating charged species from a carrier fluid and particularly to the use of a porous stationary phase that operates to reversibly remove and retain all the charged species, that can include molecules and particles, from a solution when a voltage gradient is applied to the porous stationary phase.
It is frequently the case that it is desired to analyze molecular species that are present in very low concentration either because the sample itself is very small or dilute or because the species of interest is present as a consequence of prior chemical processing and is thus at very low concentration. Moreover, these molecular species can be charged and being in the presence of uncharged molecules present further difficulty in analysis or separation.
For some time biochemists have exploited techniques wherein macromolecules are electrophoretically deposited onto porous membranes, such as nitrocellulose or porous glass. These membranes are generally impermeable to the macromolecular species and thus, this technique is essentially a form of ultrafiltration. Macromolecular species can also be concentrated by deposition on membranes by hydrostatic or centrifugal force. This same technique can be used for preconcentration of species prior to injection into a microfluidic analysis system. However, the species must be subsequently removed from the membrane prior to injection into an analysis column. Removal typically can be accomplished by the use of fluid flowing parallel to the surface of the membrane. In any case, removal by these methods can cause dilution of the species of interest, essentially negating at least part of the step of concentration.
There are numerous art-recognized techniques for concentration and separation of molecules that overcome the problems discussed above. Typical of these methods are those disclosed in U.S. Pat. No. 5,423,966 entitled xe2x80x9cOn Line Ion Contaminant Removal Apparatus and Method for Capillary Electrophoresisxe2x80x9d issued to Wiktorowicz on Jun. 13, 1995 and U.S. Pat. No. 4,617,102 entitled xe2x80x9cProcess and Apparatus for Purifying and Concentrating DNA from Crude Mixtures Containing DNAxe2x80x9d issued to Tomblin et al. Oct. 14, 1986. Here, filtration devices are used to concentrate analytes by passing a sample through a porous material. Analyte molecules smaller than the pores go through the porous structure and molecules larger than the pores are retained either within or at the upstream end of the porous material. The retained molecules can be recovered in concentrated form by reversing flow direction of the solvent or other fluid. Devices of this type retain all molecules of the size retained on the porous structure whether or not they are charged. Further, the size of the pores determine the size of the molecule trapped. If the molecule of interest is smaller than the pore size of the porous structure it cannot be retained. Thus, these methods of concentrating molecules depend upon having available a porous material that has pores of the size necessary to trap molecules of interest. Further, these methods are unable to distinguish between charged and uncharged analyte molecules.
U.S. Pat. No. 5,800,692 entitled xe2x80x9cPreseparation Processor for Use in Capillary Electrophoresisxe2x80x9d issued to Naylor et al. Sep. 1, 1998; U.S. Pat. No. 5,340,452 entitled xe2x80x9cOn-Column Preconcentration of Samples in Capillary Electrophoresisxe2x80x9d issued to Brenner et al. Aug. 23, 1994; and U.S. Pat. No. 5,453,382 entitled xe2x80x9cElectrochromatographic Preconcentration Methodxe2x80x9d issued to Novotny et al. Sep. 26, 1995 disclose another common method for concentrating an analyte, the use of a material that selectively adsorbs or binds analyte molecules allowing everything else to pass through. The adsorbed analyte can be subsequently desorbed by changing the composition of the buffer or, by way of example, by the use of electro-osmotic flow, wherein a voltage is impressed across the adsorbant to induce electro-osmotic flow thereby removing the adsorbed material from the adsorbant. Here, concentration of the analyte depends upon the selective adsorption properties of the adsorbant material consequently, these techniques generally require application-specific use of absorbent material and are unable to readily distinguish between charged or uncharged molecules. Further, desorption generally requires a change in the mobile phase or buffer.
Sample preconcentration techniques employing isotachophoresis and field-amplification in discontinuous buffer systems are disclosed in U.S. Pat. No. 5,116,471 entitled xe2x80x9cSystem and Method for Improving Sample Concentration in Capillary Electrophoresisxe2x80x9d issued to Chien et al. May 26, 1992 and U.S. Pat. No. 5,766,435 entitled xe2x80x9cConcentration of Biological Samples on a Microliter Scale and Analysis by Capillary Electrophoresisxe2x80x9d issued to Liao et al. Jun. 16, 1998. Isotachophoresis effects preconcentration of a sample by introducing a sample plug between two separate buffer systems and applying an electric field thereto. The leading electrolyte is chosen so that its mobility is faster than that of the ions in the sample while the mobility of the following electrolyte is slower. When an electric field is applied the ions order themselves according to their mobility causing the sample to be separated into zones containing its various ionic constituents. However, several problems have been encountered in the application of the method of iostachophoresis. In the absence of spacer ions, the different separated zones of a mixture border on each other and are thus difficult to recover without contamination from adjacent components. Spacer ions must possess very particular properties and thus are not always available. Moreover, to obtain adequate separation the components must be caused to move a considerable distance which demands a high voltage. Further, isotachophoresis has a limited capacity.
In field amplification schemes (commonly referred to as xe2x80x9cstackingxe2x80x9d) sample ions are introduced into a capillary column in a plug of buffer solution having a significantly lower conductivity than a background buffer electrolyte. When a voltage is applied across the capillary column the region of decreased conductivity associated with the sample experiences an increase in field strength relative to that of the background electrolyte. The increased field strength causes the charged molecules of the sample to quickly migrate to the boundary of the low conductivity zone. Crossing the boundary into the region of higher conductivity (and lower field strength) causes the charged molecules of the sample to slow down which has the effect of xe2x80x9cstackingxe2x80x9d the ions in the sample into a concentrated zone at the boundary of the two buffer regions. In order to be effective as a means of concentrating charged molecules, the method of field amplification requires precise control of both the conductivity of the background electrolyte as well as that of the sample. Thus, this method of sample concentration requires the use of two different and carefully tailored electrolytes; a common background electrolyte cannot be used.
Another method of concentrating the constituents of a sample is found in U.S. Pate. No. 4,323,439 entitled xe2x80x9cMethod and Apparatus for Dynamic Equilibrium Electrophoresisxe2x80x9d issued to O""Farrell on Apr. 6, 1982 wherein a mixture of different molecular species can be concentrated by the method of electrophoresis in the presence of a counterflowing carrier fluid in a separation chamber having a particle bed with longitudinally varying separation characteristics contained therein. This technique combines size exclusion chromatography in a packed bed of particles having a gradient of exclusion limit with countervailing electrophoresis that tends to drive the molecular species in the sample in a direction opposite that imposed by fluid flow through the chromatographic column. In this way a molecule of interest is concentrated in a zone where its convective velocity is counteracted by its electrophoretic velocity; different molecular species becoming concentrated at different locations in the column. While this method of separation and concentration of molecular species is efficient it suffers from the fact that practice requires that the column be packed with materials that provide a plurality of exclusion limits plus the need for complex flow arrangements.
Prior art provides a plurality of methods for concentrating molecular species from solution. However, there are numerous problems associated with these prior methods, such as the need for specialized column packing, the need for specialized solvents or buffer solutions, the need to change solvents or buffer solutions in order to elute concentrated molecular species, the need to change flow direction or flow conditions between the steps of retaining and eleuting species, and the inability to either separate charged and uncharged molecular species or effect an efficient separation. There exists therefore, a need for a simple way to effectively separate charged from uncharged molecules and concentrate the charged molecules from a dilute solution prior to analysis or other process steps.
The present invention discloses novel apparatus and method for controllably and quantitatively retaining and releasing charged species, including molecules and molecular aggregates such as dimers, polymers, multimers, colloids, micelles, and liposomes, contained in solution. The charged species are retained on a porous material, that can comprise the stationary phase in a column or flow channel, by applying a voltage gradient along the porous stationary phase in contact with the solution. The voltage gradient can cause the solution to move through the column by electroosmotic flow and can effect retention of charged species on the porous stationary phase while the solution is being passed through. The retained charged species can be subsequently completely removed from the porous stationary phase by applying a pressure differential to the column, preferably in the substantial absence of the applied voltage gradient. Thus, the present invention provides for separating charged molecules from uncharged or neutral molecules, irrespective of the size or shape difference, and for concentrating charged molecules from dilute solution using reversible electric field induced retention of charged molecules in volumes and on surfaces of porous materials. In the inventive method, uncharged molecules pass through the system unimpeded thus effecting a complete separation of charged and uncharged molecules and making possible concentration factors as least as great as 1000-fold. Moreover, the pressure driven flow that removes the retained charged molecules from the porous matrix is independent of direction and thus neither means to reverse fluid flow nor a multi-directional flow field is required-a single flow through bed can be employed in contrast to prior art systems.